DISPATCHES recovered during the summer of 2002 in stools of an HIV- Cryptosporidium positive Peruvian woman with severe diarrhea. This find- ing was confirmed by light microscopy, polymerase chain muris, a Rodent reaction (PCR)–restriction fragment length polymorphism Pathogen, (RFLP), and DNA sequencing. The Study Recovered from a In 2002, we conducted a year-long collaborative study on the epidemiology of Cyclospora cayetanensis infec- Human in Perú tions in Perú. As part of that study, we collected approxi- mately 100 stool samples in 2.5% potassium dichromate Carol J. Palmer,* Lihua Xiao,† solution from persons in Lima and Iquitos with Angélica Terashima,‡ Humberto Guerra,‡ Cyclospora infection. Fecal samples were initially identi- Eduardo Gotuzzo,‡ Gustavo Saldías,§ fied as Cyclospora-positive in Lima, and then transported J. Alfredo Bonilla,* Ling Zhou,† Alan Lindquist,¶ to the United States for additional confirmation using wet and Steve J. Upton# mount and Nomarski interference contrast microscopy. Cryptosporidium muris, predominantly a rodent Two stool samples, which were taken on two sequential species of Cryptosporidium, is not normally considered a days from an HIV-positive woman who was 31 years of human pathogen. Recently, isolated human infections have age, contained oocysts that appeared, based on morpholo- been reported from Indonesia, Thailand, France, and gy, to be Cryptosporidium muris. Low numbers of Kenya. We report the first case of C. muris in a human in Cyclospora cayetanensis and Blastocystis hominis oocysts the Western Hemisphere. This species may be an emerg- were also identified in the stool samples. The ing zoonotic pathogen capable of infecting humans. Cryptosporidium muris infection was initially identified by using wet mount microscopy with oocysts (n=25) aver- ryptosporidiosis can be a debilitating diarrheal dis- aging 6.1 (± 0.3) x 8.4 (± 0.3) µM (range 5.6–6.4 x Cease. While infections are normally acute and self- 8.0–9.0) and a shape index (length/width) 1.38 (1.25–1.61) limiting in immunocompetent persons, cryptosporidiosis (Figure 1). Numbers of oocysts were determined semi- can be life threatening in those with compromised immune quantitatively in each sample by hemacytometer, with an systems. In humans, cryptosporidiosis is caused predomi- estimated 737,000 and 510,000 oocysts/g recovered from nantly by Cryptosporidium parvum or C. hominis (the lat- the submitted samples on day 1 and day 2, respectively. ter was previously known as the C. parvum human geno- The diagnosis of C. muris was later confirmed through type), and major outbreaks of the disease have been clear- DNA analysis. ly associated with contaminated drinking water (1). HIV was first diagnosed in the patient in November Recently, another species of Cryptosporidium, C. 2000 by using enzyme-linked immunosorbent assay and muris, has been suggested to be of concern to human Western blot (immunoblot). She arrived at the hospital health. C. muris is a parasite first identified in the gastric clinic in June 2002 with fever and reported that she had glands of mice (2). Experimental transmission studies have been experiencing diarrhea for >3 months. The patient shown that the parasite readily infects multiple nonrodent reported that she had lost approximately 25 lbs. in the past hosts including dogs, rabbits, lambs, and cats (3). C. 7 months, consistent with HIV-wasting syndrome. Her muris–like organisms have also been reported as oppor- chest x-ray was abnormal, but four direct sputum examina- tunistic infectious agents in immunocompromised nonhu- tions for acid-fast bacteria using Ziehl-Neelsen staining man primates (4). In the past 2 years, five cases of infec- were negative, as were efforts at culturing Mycobacterium tions with C. muris or C. muris–like parasites have been tuberculosis. reported from HIV-positive and healthy persons in Kenya Other laboratory values for this patient at the time of (5), France (6), Thailand (7), and Indonesia (8). In this stool sample collection were as follows: CD4 cell count paper, we report on the first documented case of C. muris 66/µL; hematocrit 36%; leukocytes 4,100/µL with 4% in a human in the Western Hemisphere. The parasite was bands, 55% neutrophils, 27% lymphocytes, and 0% eosinophils; urine examination normal; creatine 0.8 *University of Florida, Gainesville, Florida, USA; †Centers for mg/dL; urea 21 mg/dL; glucose 105 mg/dL; serum glutam- Disease Control and Prevention, Atlanta, Georgia, USA; ic oxalacetic transaminase 30 IU/L; serum glutamic pyru- ‡Universidad Peruana Cayetano Heredia, Lima, Perú; §London vic transaminase 46 IU/L; and bilirubin 0.9 mg/dL. School of Tropical Medicine, London, United Kingdom; ¶United The diagnosis of Cryptosporidium in the patient’s sam- States Environmental Protection Agency, Cincinnati, Ohio, USA; #Kansas State University, Manhattan, Kansas, USA ples was confirmed by a small subunit rRNA-based nested 1174 Emerging Infectious Diseases • Vol. 9, No. 9, September 2003 DISPATCHES molecular tools (8). One putative C. muris infection was reported in an immunocompromised patient in France based on sequence analysis of a small fragment of the SSU rRNA (6). However, the sequence presented was more similar to that of C. andersoni (2-bp differences in a 242- bp region) than to C. muris (8-bp differences in the region). Although determining whether or not the C. muris con- tributed medical problems in this patient is not possible, detecting C. muris in her stool sample is an unexpected Figure 1. Nomarski interference contrast photomicrographs of finding. A major difference between C. parvum or C. Cryptosporidium muris from the feces of an HIV-positive human. hominis and C. muris, is that C. parvum and C. hominis Scale bars = 5 µm. normally colonize the intestine, whereas C. muris is a gas- tric pathogen in cattle. Anderson (12) and Esteban and PCR, which amplified a portion of the rRNA gene (830 Anderson (13) reported that another gastric species, C. bp). Cryptosporidium spp. was determined by the banding andersoni, infects only the glands of the cattle stomach patterns of restriction digestions of PCR products with (abomasum), where it retards acid production. These SspI, VspI, and DdeI (9). Diagnosis was confirmed by researchers postulated that this process may affect protein DNA sequencing of three independent PCR products from digestion in the abomasum and account for the fact that each sample in both directions on an ABI PRISM 3100 milk production in cows that are chronically infected with (Applied Biosystems, Foster City, CA) instrument. Figure C. muris appears to be reduced by approximately 13%. 2 shows the RFLP analysis of three PCR products from Thus, an infection by C. muris may perhaps cause similar each sample with restriction enzymes SspI and VspI; these protein digestion problems in human infections, particular- results suggest that these PCR products belonged to either ly in HIV-positive persons. C. muris or Cryptosporidium andersoni (10). Further Even though only a few cases of C. muris infections RFLP analysis with DdeI showed banding patterns identi- have been identified so far in humans, gastric cryp- cal to C. muris (9; Figure 2). All DNA sequences obtained tosporidiosis occurs much more often than believed, espe- from the six PCR products were identical to those previ- cially in HIV-positive persons. Up to 40 % of cryp- ously reported by Xiao et al. (10,11) from C. muris from a tosporidiosis in HIV-infected persons includes gastric Bactrian camel, a rock hyrax, and mice (GenBank acces- involvement (14). Although most gastric Cryptosporidium sion nos. AF093997 and AF093498) and another isolate infections in HIV-positive persons are likely caused by C. recently found in an HIV patient in Kenya (5). parvum or C. hominis because of immunosuppression, the After the diagnosis of intestinal parasite infection, the contribution of C. muris probably has been underestimat- patient was treated with TMP-SMX (trimethoprim 160 ed. Thus, molecular characterizations of stomach tissues mg, sulfamethoxazole 800 mg) Forte twice a day for 1 from patients with gastric cryptosporidiosis may help us to week and then TMP-SMX once a day for Pneumocystis understand the pathogenesis of human Cryptosporidium carinii pneumonia prophylaxis. The patient was also infection. placed on AZT/3TC and nevirapine. The patient recovered with no further evidence of Cyclospora, Blastocystis, or C. muris in stool samples taken 2 months posttreatment. She became afebrile and had gained 5 kg as of 2 months’ post- treatment. Molecular analysis of a stool sample collected 122 days after the initial diagnosis confirmed that the patient had recovered from the C. muris infection. Conclusions This report represents the third confirmed case of C. muris infection in humans. Previously, one case of C. muris infection was identified in an HIV-positive child in Figure 2. Identification of Cryptosporidium muris from two stool Thailand and in an HIV-positive adult in Kenya using samples from a Peruvian patient using restriction fragment length microscopy and molecular analysis (5,7). C. muris and C. polymorphism analysis of polymerase chain reaction products with SspI (A), VspI (B) and DdeI (C). Lane 1, 100-bp molecular mark- andersoni–like oocysts were found in two healthy ers; lane 2, C. hominis control; lanes 3 and 4, C. muris from the Indonesian girls, but the diagnosis was not confirmed by patient. Emerging Infectious Diseases • Vol. 9, No. 9, September 2003 1175 DISPATCHES Our report expands the geographic range of suspect C. 3. Izeki M, Maekawa T, Moriya K, Uni S, Takada S. Infectivity of muris infections in humans and suggests that this species Cryptosporidium muris (strain RN 66) in various laboratory animals. Parasitol Res 1989;75:218–22. may be a global emerging zoonotic pathogen. This 4. Dubey JP, Markovitis JE, Killary KA. Cryptosporidium muris–like pathogen may be of particular importance to persons living infection in stomach of cynomolgus monkeys (Macaca fascicularis).